The goal of this research is to reveal the mechanistic basis and evolutionary dynamics by which specific functional interactions between adrenal steroids and their receptors evolved. An interdisciplinary approach combining molecular endocrinology, phylogenetics, and evolutionary analysis will be used to identify the amino acid changes that conferred on the glucocorticoid and mineralocorticoid receptors (GR and MR) their specific activation by cortisol and aldosterone and to characterize the dynamics of the process by which these functions evolved. This will entail identification of "stem" receptors that diverged from the family before the GR and MR evolved: steroid receptors from the hagfish, a key phylogentic species due to its position near the base of the vertebrate lineage, will be isolated and functionally analyzed, as will those from another basal vertebrate, the sea lamprey. The steroid receptor family phylogeny will then be inferred, providing a scaffold on which to infer the dynamics and reconstruct the specific evolutionary changes by which they have diversified in number, function, sequence, and structure. State-of-the-art statistical techniques will be used to reconstruct the sequence of the ancestral receptor from which the MR and GR diverged, and this ancient gene will be synthesized, expressed in cell culture, and functionally characterized to determine which functions are ancestral and derived. Hypotheses about the mechanistic basis for the emergence of novel functions will then be generated based on the phylogenetic reconstruction and extant structure-function data, and these hypotheses will be tested by introducing specific mutations of putative functional importance into both ancestral and extant receptors. These sites are predicted to provide ideal targets for therapeutic interventions for heart and kidney disease.